人参总皂苷诱导宫颈癌HeLa细胞凋亡和自噬

Total Ginsenosides Extract Induces Apoptosis and Autophagy of Cervical Cancer HeLa Cells

目的探讨人参总皂苷(total ginsenosides extract, TGS)对宫颈癌HeLa细胞凋亡和自噬的影响。方法应用不同浓度的TGS作用HeLa细胞24 h,采用CCK-8法检测TGS对宫颈癌HeLa细胞增殖的影响并筛选出最佳浓度;Annexin V-FITC/PI双染流式细胞术检测细胞凋亡;通过GFP-LC3质粒转染HeLa细胞并加入TGS观察LC3斑点的数量,以及吖啶橙染色后观察自噬溶酶体的形成情况。蛋白质免疫印迹分析法检测细胞中凋亡相关蛋白和自噬相关蛋白的表达水平,对TGS诱导的凋亡和自噬进行验证。结果 CCK-8法显示TGS显著了抑制HeLa细胞增殖,且具有浓度依赖性(P<0.05);流式细胞仪检测结果表明,TGS处理后HeLa细胞凋亡率明显升高;GFP-LC3质粒转染分析结果显示,TGS在HeLa细胞中明显增加了LC3斑点的数量;吖啶橙染色后观察到细胞内自噬溶酶体明显增多;蛋白质免疫印迹分析法结果表明与对照组比较,TGS各剂量细胞中凋亡抑制因子Bcl-2表达水平降低,凋亡促进因子Bax表达水平升高;自噬相关因子p-AKT/AKT、p-mTOR/mTOR比值逐渐降低,LC3B-Ⅱ和Beclin-1的表达水平均明显上调,且差异有统计学意义(P<0.05)。结论人参总皂苷显著抑制宫颈癌HeLa细胞增殖并具有剂量依赖性,促进其发生细胞凋亡并引起AKT/mTOR途径相关的细胞自噬。

Objective To investigate the effects of total ginsenosides extract (TGS) on apoptosis and autophagy in cervical cancer HeLa cells. Methods HeLa cells were treated with different concentrations of TGS for 24 h. The effect of TGS on the proliferation of cervical cancer HeLa cells was detected by CCK-8 method and the optimal concentration was screened. Apoptosis was detected by Annexin V-FITC/PI double staining flow cytometry. HeLa cells were transfected with the GFP-LC3 plasmid and TGS was added to observe the number of LC3 spots, and the formation of autophagolysosomes was observed after acridine orange staining. Western blot analysis was used to detect the expression levels of apoptosis-related proteins and autophagy-related proteins in cells to verify the TGS-induced apoptosis and autophagy. Results CCK-8 method showed that TGS significantly inhibited HeLa cells proliferation in a concentration-dependent manner (P<0.05). The results of flow cytometry showed that the apoptosis rate of HeLa cells increased significantly after TGS treatment. The results of GFP-LC3 plasmid transfection analysis showed that TGS significantly increased the number of LC3 spots in HeLa cells. After acridine orange staining, a significant increase of autophagy lysosomes in intracellular was observed. Western blot analysis showed that compared with that of the control group, the expression of apoptosis inhibitor Bcl-2 decreased and the expression of apoptosis promoter Bax increased in TGS cells at different doses. The ratios of autophagy-related factors p-AKT/AKT and p-mTOR/mTOR were gradually decreased, and the expression levels of LC3 B-Ⅱand Beclin-1 were significantly increased, and the difference was statistically significant (P<0.05). Conclusion TGS significantly inhibited the proliferation of cervical cancer HeLa cells in a dose-dependent manner, promoted their apoptosis and caused autophagy in cells related to the AKT/mTOR pathway.